Structural investigations of stereoselective profen binding by equine and leporine serum albumins

Serum albumin, the most abundant transport protein of mammalian blood, interacts with various nonsteroidal anti-inflammatory drugs (NSAIDs) affecting their disposition, metabolism, and excretion. A big group of chiral NSAIDs transported by albumin, profens, is created by derivatives of 2-arylpropionic acid. The chiral center in the structures of profens is adjacent to the carboxylate moiety and often determines different pharmacological properties of profen enantiomers. This study describes crystal structures of two albumins, isolated from equine and leporine serum, in complexes with three profens: ibuprofen, ketoprofen, and suprofen. Based on three-dimensional structures, the stereoselectivity of albumin is discussed and referred to the previously published albumin complexes with drugs. Drug Site 2 (DS2) of albumin, the bulky hydrophobic pocket of subdomain IIIA with a patch of polar residues, preferentially binds (S)-enantiomers of all investigated profens. Almost identical binding mode of all these drugs clearly indicates the stereoselectivity of DS2 towards (S)-profens in different albumin species. Also, the affinity studies show that DS2 is the major site that presents high affinity towards investigated drugs. Additionally, crystallographic data reveal the secondary binding sites of ketoprofen in leporine serum albumin and ibuprofen in equine serum albumin, both overlapping with previously identified naproxen binding sites: the cleft formed between subdomains IIIA and IIIB close to the fatty acid binding site 5 and the niche created between subdomains IIA and IIIA, called fatty acid site 6.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Investigations on the interactions of DiAmsar with serum albumins: Insights from spectroscopic and molecular docking techniques

Diamine‐sarcophagine (DiAmsar) binding to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under simulative physiological conditions. Fluorescence spectra in combination with Fourier transform infrared (FT‐IR), UV‐visible (UV–vis) spectroscopy, cyclic voltammetry (CV), and molecular docking method were used in the present work. Experimental results revealed that DiAmsar had an ability to quench the HSA and BSA intrinsic fluorescence through a static quenching mechanism. The Stern–Volmer quenching rate constant (K_sv) was calculated as 0.372 × 10³ M^‐1 and 0.640 × 10³ M^‐1 for HSA and BSA, respectively. Moreover, binding constants (K_a), number of binding sites (n) at different temperatures, binding distance (r), and thermodynamic parameters (?H°, ?S°, and ?G°) between DiAmsar and HSA (or BSA) were calculated. DiAmsar exhibited good binding propensity to HSA and BSA with relatively high binding constant values. The positive ?H° and ?S° values indicated that the hydrophobic interaction is main force in the binding of the DiAmsar to HSA (or BSA). Furthermore, molecular docking results revealed the possible binding site and the microenvironment around the bond. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies on the synthesis,characterization, human serum albumin binding and biological activity of single chain surfactant–cobalt(III) complexes

The interaction of surfactant–cobalt(III) complexes [Co(bpy)(dien)TA](ClO₄)₃ · 3H₂O (1) and [Co(dien)(phen)TA](ClO₄)₃ · 4H₂O (2), where bpy = 2,2′‐bipyridine, dien = diethylenetriamine, phen = 1,10‐phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (K_b) and number of binding‐sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (?G°, ?H° and ?S°) and E_a were also obtained. According to Förster's non‐radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the interactions of 3,6‐diaminoacridine derivatives with human serum albumin by fluorescence spectroscopy

This study reports the preparation and investigation of the modes of binding of the two symmetric 3,6‐diaminoacridine derivatives obtained from proflavine, which are 3,6‐diphenoxycarbonyl aminoacridine and 3,6‐diethoxycarbonyl aminoacridine to human serum albumin (HSA). The interaction of HSA with the derivatives was investigated using fluorescence quenching and ultraviolet‐visible absorption spectra at pH 7.2 and different temperatures. The results suggest that the derivatives used can interact strongly with HSA and are the formation of HSA‐derivative complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern‐Volmer quenching constants, binding constants, binding sites and corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The binding distance (r) ~ 3 nm between the donor (HSA) and acceptors (3,6‐diethoxycarbonyl aminoacridine, 3,6‐diphenoxycarbonyl aminoacridine and proflavine) was obtained according to Förster's non‐radiative energy transfer theory. Moreover, the limit of detection and limit of quantification of derivatives were calculated in the presence of albumin. Copyright © 2014 John Wiley & Sons, Ltd.     

Behavior of various mammalian albumins towards bilirubin binding and photochemical properties of different bilirubin-albumin complexes

Bilirubin (BR) binding properties of serum albumins from different mammalian species viz. human (HSA), equine (ESA), dog (DSA) and guinea pig (GPSA) were studied by absorption, fluorescence and CD spectroscopy. Whereas, a complex of BR with ESA produced maximum change, GPSA–BR complex showed weaker interaction as reflected from absorption and fluorescence spectroscopic data. Conformational analysis of these albumins by near- and far-UV CD spectra suggested similar structural characteristics (both secondary and tertiary structures) for ESA and HSA, whereas, DSA and GPSA had lower amounts of secondary and tertiary structures being minimum for GPSA. Photoirradiation results of BR–albumin complexes showed GPSA-bound BR more labile compared with other complexes, whereas, BR–ESA complex was found to be more stable against photoinduced chemical changes. Taken together, all these results suggest that chiroptical properties/stability of albumin bound BR varies with albumin species.     

Exploration of human serum albumin binding sites by docking and molecular dynamics flexible ligand–protein interactions

Five‐nanosecond molecular dynamics (MD) simulations were performed on human serum albumin (HSA) to study the conformational features of its primary ligand binding sites (I and II). Additionally, 11 HSA snapshots were extracted every 0.5 ns to explore the binding affinity (K_d) of 94 known HSA binding drugs using a blind docking procedure. MD simulations indicate that there is considerable flexibility for the protein, including the known sites I and II. Movements at HSA sites I and II were evidenced by structural analyses and docking simulations. The latter enabled the study and analysis of the HSA–ligand interactions of warfarin and ketoprofen (ligands binding to sites I and II, respectively) in greater detail. Our results indicate that the free energy values by docking (K_d observed) depend upon the conformations of both HSA and the ligand. The 94 HSA–ligand binding K_d values, obtained by the docking procedure, were subjected to a quantitative structure‐activity relationship (QSAR) study by multiple regression analysis. The best correlation between the observed and QSAR theoretical (K_d predicted) data was displayed at 2.5 ns. This study provides evidence that HSA binding sites I and II interact specifically with a variety of compounds through conformational adjustments of the protein structure in conjunction with ligand conformational adaptation to these sites. These results serve to explain the high ligand‐promiscuity of HSA. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 161–170, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic,cyclic voltammetric,and molecular docking technique

The interaction of fisetholz with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. The results revealed that there was a static quenching of BSA/HSA induced by fisetholz. The binding constants (K_a) and binding sites (n) were calculated at different temperatures (293, 303, and 311?K). The enthalpy change (ΔH) were calculated to be –17.20?kJ mol^?1 (BSA) and –18.28?kJ mol^?1 (HSA) and the entropy change (ΔS) were calculated to be 35.41?J mol^?1 (BSA) and 24.02?J mol^?1 (HSA), respectively, which indicated that the interaction between fisetholz and BSA/HSA was mainly by electrostatic attraction. Based on displacement experiments using site probes, indomethacin and ibuprofen, the binding site of fisetholz to BSA/HSA was identified as sub-domain IIIA, which was further confirmed by molecular docking method. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ on fisetholz-BSA or fisetholz-HSA complex. The spectra of synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) all showed that fisetholz binding to BSA/HSA leads to secondary structures change of the two serum albumins. According to the Förster non-radiation energy transfer theory, the binding distance between fisetholz and BSA/HSA was 2.94/4.68?nm. The cyclic voltammetry as a supporting tool also indicated that fisetholz interacted with protein.

Communicated by Ramaswamy H. Sarma     

The interaction between cepharanthine and two serum albumins: multiple spectroscopic and chemometric investigations

The binding modes of cepharanthine (CEPT) with bovine serum albumin (BSA) and human serum albumin (HSA) have been established by reproducing physiological conditions, which is very important to understand the pharmacokinetics and toxicity of CEPT. These spectral data were further analyzed by the multivariate curve resolution‐alternating least squares method. Moreover, the concentration profiles and pure spectra of three species (BSA/HSA, CEPT and CEPT–BSA/HSA) and the apparent equilibrium constants K_app were evaluated. The experimental results showed that CEPT could quench the fluorescence intensity of BSA/HSA by a combined quenching (static and dynamic) procedure. The binding constant (K), the thermodynamic parameters (ΔG, ΔH and ΔS) and binding subdomain were measured, and indicated that CEPT could spontaneously bind to BSA/HSA on subdomain IIA through the hydrophobic interactions. The effect of CEPT on the secondary structure of proteins has been analyzed by circular dichroism, 3D fluorescence and Fourier transform infrared spectra. The binding distance between CEPT and tryptophan of BSA/HSA was 2.305/1.749 nm, which is based on the Förster resonance energy transfer theory. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis and application of thiol‐functionalized magnetic nanoparticles for studying interactions of epirubicin hydrochloride with bovine serum albumin by fluorescence spectrometry

A novel method was developed for studying the interaction between epirubicin hydrochloride (EPI) and bovine serum albumin (BSA) by fluorescence spectrometry. Fe₃O₄ magnetic nanoparticles (MNPs) synthesized and functionalized with thiol group were employed for the immobilization and separation of target BSA in reaction solutions. The concentrations of the non‐immobilized BSA and unbound EPI were obtained separately by fluorescence spectrometry. The binding constants (K _a ) and number of binding sites (n ) of EPI with BSA were calculated. In this study, the K _a value was 5.05 × 10⁵ L mol^?1, suggesting a strong binding of EPI to BSA, and the n value was 1.15. The effects of common metal ions on K _a of EPI with BSA were also investigated, and the results showed there was clearly bindings between the metal ions and BSA. The precise binding sites of EPI on BSA were determined as being in site I from the competitive displacement experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Exploring the interactions of a Tb(III)–quercetin complex with serum albumins (HSA and BSA): spectroscopic and molecular docking studies

Serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA), two main circulatory proteins), are globular and monomeric macromolecules in plasma that transport many drugs and compounds. In the present study, we investigated the interactions of the Tb(III)–quercetin (Tb–QUE) complex with HSA and BSA using common spectroscopic techniques and a molecular docking study. Fluorescence data revealed that the inherent fluorescence emission of HSA and BSA was markedly quenched by the Tb–QUE complex through a static quenching mechanism, confirming stable complex formation (a ground‐state association) between albumins and Tb–QUE. Binding and thermodynamic parameters were obtained from the fluorescence spectra and the related equations at different temperatures under biological conditions. The binding constants (K_b) were calculated to be 0.8547 × 10³ M^?1 for HSA and 0.1363 × 10³ M^?1 for BSA at 298 K. Also, the number of binding sites (n) of the HSA/BSA–Tb–QUE systems was obtained to be approximately 1. Thermodynamic data calculations along with molecular docking results indicated that electrostatic interactions have a main role in the binding process of the Tb–QUE complex with HSA/BSA. Furthermore, molecular docking outputs revealed that the Tb–QUE complex has high affinity to bind to subdomain IIA of HSA and BSA. Binding distances (r) between HSA–Tb–QUE and BSA–Tb–QUE systems were also calculated using the Forster (fluorescence resonance energy transfer) method. It is expected that this study will provide a pathway for designing new compounds with multiple beneficial effects on human health from the phenolic compounds family such as the Tb–QUE complex.     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction of cyproheptadine hydrochloride with human serum albumin using spectroscopy and molecular modeling methods

The interaction between cyproheptadine hydrochloride (CYP) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT‐IR) and molecular modeling at a physiological pH (7.40). Fluorescence of HSA was quenched remarkably by CYP and the quenching mechanism was considered as static quenching since it formed a complex. The association constants K_a and number of binding sites n were calculated at different temperatures. According to Förster's theory of non‐radiation energy transfer, the distance r between donor (human serum albumin) and acceptor (cyproheptadine hydrochloride) was obtained. The effect of common ions on the binding constant was also investigated. The effect of CYP on the conformation of HSA was analyzed using FT‐IR, synchronous fluorescence spectroscopy and 3D fluorescence spectra. The thermodynamic parameters ΔH and ΔS were calculated to be ?14.37 kJ mol^?1 and 38.03 J mol^?1 K^?1, respectively, which suggested that hydrophobic forces played a major role in stabilizing the HSA‐CYP complex. In addition, examination of molecular modeling indicated that CYP could bind to site I of HSA and that hydrophobic interaction was the major acting force, which was in agreement with binding mode studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies on binding interactions between clenbuterol hydrochloride and two serum albumins by multispectroscopic approaches in vitro

In this study, binding properties of clenbuterol hydrochloride (CL) with human serum albumin (HSA) and bovine serum albumin (BSA) were examined using constant protein concentrations and various CL contents under physiological conditions. The binding parameters were confirmed using fluorescence quenching spectroscopy at various temperatures. The experimental results confirmed that the quenching mechanisms of CL and HSA/BSA were both static quenching processes. The thermodynamic parameters, namely, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to the van't Hoff equation, which suggested that the electrostatic interactions were the predominant intermolecular forces in stabilizing the CL–HSA complex, and hydrogen bonds and van der Waals force were the predominant intermolecular forces in stabilizing the CL–BSA complex. Furthermore, the conformational changes of HSA/BSA in the presence of CL were determined using the data obtained from three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy and circular dichroism spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Binding of hydroxylated polybrominated diphenyl ethers with human serum albumin: Spectroscopic characterization and molecular modeling

Three hydroxylated polybrominated diphenyl ethers (OH‐PBDEs), 3‐OH‐BDE‐47, 5‐OH‐BDE‐47, and 6‐OH‐BDE‐47, were selected to investigate the interactions between OH‐PBDEs with human serum albumin (HSA) under physiological conditions. The observed fluorescence quenching can be attributed to the formation of complexes between HSA and OH‐PBDEs. The thermodynamic parameters at different temperatures indicate that the binding was caused by hydrophobic forces and hydrogen bonds. Molecular modeling and three‐dimensional fluorescence spectrum showed conformational and microenvironmental changes in HSA. Circular dichroism analysis showed that the addition of OH‐PBDEs changed the conformation of HSA with a minor reduction in α‐helix content and increase in β‐sheet content. Furthermore, binding distance r between the donor (HSA) and acceptor (three OH‐PBDEs) calculated using Förster's nonradiative energy transfer theory was <7 nm; therefore, the quenching mechanisms for the binding between HSA and OH‐PBDEs involve static quenching and energy transfer. Combined with molecular dynamics simulations, the binding free energies (ΔG _bind ) were calculated using molecular mechanics/Poisson ? Boltzmann surface area method, and the crucial residues in HSA were identified.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

Interactive association between RhoA transcriptional signaling inhibitor,CCG1423 and human serum albumin: Biophysical and in silico studies

Multiple spectroscopic techniques, such as fluorescence, absorption, and circular dichroism along with in silico studies were used to characterize the binding of a potent inhibitor molecule, CCG1423 to the major transport protein, human serum albumin (HSA). Fluorescence and absorption spectroscopic results confirmed CCG1423–HSA complex formation. A strong binding affinity stabilized the CCG1423–HSA complex, as evident from the values of the binding constant (K_a = 1.35 × 10⁶–5.43 × 10⁵ M^?1). The K_SV values for CCG1423–HSA system were inversely correlated with temperature, suggesting the involvement of static quenching mechanism. Thermodynamic data anticipated that CCG1423–HSA complexation was mainly driven by hydrophobic and van der Waals forces as well as hydrogen bonds. In silico analysis also supported these results. Three-dimensional fluorescence and circular dichroism spectral analysis suggested microenvironmental perturbations around protein fluorophores and structural (secondary and tertiary) changes in the protein upon CCG1423 binding. CCG1423 binding to HSA also showed some protection against thermal denaturation. Site-specific marker-induced displacement results revealed CCG1423 binding to Sudlow’s site I of HSA, which was also confirmed by the computational results. A few common ions were also found to interfere with the CCG1423–HSA interaction.     

Multispectroscopic exploration and molecular docking analysis on interaction of eriocitrin with bovine serum albumin

Eriocitrin is a flavanone glycoside, which exists in lemon or lime citrus fruits. It possesses antioxidant, anticancer, and anti‐allergy activities. In order to investigate the pharmacokinetics and pharmacological mechanisms of eriocitrin in vivo, the interaction between eriocitrin and bovine serum albumin (BSA) was studied under the simulated physiological conditions by multispectroscopic and molecular docking methods. The results well indicated that eriocitrin and BSA formed a new eriocitrin‐BSA complex because of intermolecular interactions, which was demonstrated by the results of ultraviolet‐visible (UV‐vis) absorption spectra. The intrinsic fluorescence of BSA was quenched by eriocitrin, and static quenching was the quenching mechanism. The number of binding sites (n) and binding constant (K_b) at 310 K were 1.22 and 2.84 × 10⁶ L mol^?1, respectively. The values of thermodynamic parameters revealed that the binding process was spontaneous, and the main forces were the hydrophobic interaction. The binding distance between eriocitrin and BSA was 3.43 nm. In addition, eriocitrin changed the conformation of BSA, which was proved by synchronous fluorescence and circular dichroism (CD) spectra. The results of site marker competitive experiments suggested that eriocitrin was more likely to be inserted into the subdomain IIA (site I), which was further certified by molecular docking studies.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996